Cell viability for SK‐Mel 28 (left panel) and SK‐Mel 24 (right panel) was measured after 72‐h treatment with other Hsp90 inhibitors (aside from XL888), such as AUY022, BIIB021, novobiocin, and 17‐DMAG. (Discrepancies in the concentration–response profiles for SK‐Mel 28 to XL888 between (B and D) can be attributed to the viability surrogate that is measured. In the case of (B), the MTS assay, quantifying metabolic activity, is used, whereas (D) is based on readout of ATP using CellTiter‐Glo. In both assays, incomplete killing occurs at the concentrations of drugs used) (±SD is plotted; n =3).

Protein interaction map built using Cytoscape 3.2 and Reactome as plugin (see Appendix) and the statistical significant entries generated from the comparison of the proteome and phosphoproteome of drug versus DMSO for sensitive and resistant cells in both “lysate” and “intact cell” layouts.

Protein interaction map built using Cytoscape 3.2 and Reactome as plugin (see Appendix) and the statistical significant entries generated from the comparison of the proteome and phosphoproteome thermal stability of resistant versus sensitive cells different settings.

Western blots of protein expression levels of CDK2 in A375 DR1, ESTDAB 37, M026.X1.CL, and MNT‐1 DR100 upon treatment with DMSO, BRAFi, and Hsp90i at 72 h (left panel). Band intensities for the quantification of CDK2 expression levels in different cell lines in different settings were normalized against the mean of GAPDH, and DMSO treatment was used as reference (right panel).

Western blot of protein expression levels of AKT1 in SK‐Mel 24 and SK‐Mel 28 in different settings at 48‐h treatment.

The same rationale used in (A) was exploited for the proteomics analysis of the effects of BRAFi (1 μM dabrafenib) treatment (upper panel) after 48 h (n = 3). Volcano plot generated by the comparison between BRAFi/DMSO in SK‐Mel 28 after 48 h (n = 3; lower panel).

The same rationale used in (A) was exploited for the proteomics analysis of BRAFi‐Hsp90i combined therapy after 48 h (n = 3).

Overlap of the downregulated (upper panel) and upregulated (lower panel) kinases at proteomics level unique for SK‐Mel 28 in different settings at 48 h (n = 3). In red, the only shared “druggable” upregulated kinase CDK2 is highlighted.

Western blot analysis confirms the upregulation of CDK2 in different settings (upper panel). Band intensities were normalized against the mean of β‐actins, and lane 1 was used as reference (lower panel).

Western blot analyses confirmed our bioinformatics prediction analyses by ChEA. Note the inverse correlation between pERK and MITF expressions in SK‐Mel 28. The color of the cell pellets of SK‐Mel 28 upon treatments at 48 h with DMSO, BRAFi, and Hsp90i is shown in the lower panel.

The knockdown of MITF causes downregulation of CDK2 expression levels in SK‐Mel 28.